The elution profile of immobilized liposome chromatography: determination of association and dissociation rate constants.

The interaction of lipophilic cations, tetraphenylphosphonium and triphenylphosphonium homologues with liposomes was investigated using immobilized liposome chromatography (ILC). Large unilamellar liposomes with a mean diameter of 100 nm were stably immobilized in chromatographic gel beads by avidin–biotin. The distribution coefficient calculated from (Ve−V0)/Vs (Ve, retention volume; V0, the void volume; Vs, the stationary phase volume) was found to be independent of flow rate, injection amount and gel bed volume, which is consistent with chromatograph theory. The relationship between the bandwidth and solvent flow rate did not follow band-broadening theories reported thus far. We hypothesized that the solvent might be forced to produce large eddies, spirals or turbulent flow due to the presence of liposomes fixed in the gel. Therefore, we developed a new theory for ILC elution: The column is composed of a number of thin disks containing liposomes and solution, and within each disk the solution is well mixed. This theory accounts for our results, and we were able to use it to estimate the rate constants of association and dissociation of the phosphonium to/from liposomes

Application of fluorescence resonance energy transfer (FRET) to investigation of light-induced conformational changes of the phoborhodopsin/transducer complex

The photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) forms a complex with its cognate the Halobacterial transducer II (pHtrII) in the membrane, through which changes in the environmental light conditions are transmitted to the cytoplasm in Natronomonas pharaonis to evoke negative phototaxis. We have applied a fluorescence resonance energy transfer (FRET)- based method for investigation of the light-induced conformational changes of the ppR/pHtrII complex. Several far-red dyes were examined as possible fluorescence donors or acceptors because of the absence of the spectral overlap of these dyes with all the photointermediates of ppR. The flash-induced changes of distances between the donor and an acceptor linked to cysteine residues which were genetically introduced at given positions in pHtrII(1-159) and ppR were determined from FRET efficiency changes. The dye-labeled complex was studied as solubilized in 0.1% n-dodecyl-β-D-maltoside (DDM). The FRET-derived changes in distances from V78 and A79 in pHtrII to V185 in ppR were consistent with the crystal structure data [Moukhametzianov, R. et al. (2006) Nature, 440, 787-792]. The distance from D102 in pHtrII linker region to V185 in ppR increased by 0.33Å upon the flash excitation. These changes arose within 70ms (the dead time of instrument) and decayed with a rate of 1.1±0.2s. Thus, sub angstrom-scale distance changes in the ppR/pHtrII complex were detected with this FRET-based method using far-red fluorescent dyes; this method should be a valuable tool to investigate conformation changes in the transducer, in particular its dynamics

A stable association with PME‐1 may be dispensable for PP2A demethylation – implications for the detection of PP2A methylation and immunoprecipitation

Reversible methyl‐esterification (methylation) of Leu309 in the protein phosphatase 2A catalytic subunit (PP2Ac) is essential for proper biogenesis of the PP2A holoenzyme. Accumulating evidence links PP2Ac methylation to diseases, including cancer and neurodegenerative disorders. Protein phosphatase methyl‐esterase (PME‐1) specifically catalyzes PP2Ac demethylation. We demonstrate that PP2Ac is demethylated in cell extracts even at 0 °C unless prevented by a PME‐1 methyl‐esterase inhibitor. This promotes dissociation of PP2A heterotrimers with B55 or PR72 subunits, but not those with B56 subunits. These results reveal differential sensitivity of ABC heterotrimers to methylation status of the C subunit. Our study advocates caution when interpreting earlier findings, offers an effective protocol for preserving PP2A complexes, and reveals key distinctions between B subunits and their interactions with the AC core dimer of PP2A

Detection of a High-Density Brachiolaria-Stage Larval Population of Crown-of-Thorns Sea Star (Acanthaster planci) in Sekisei Lagoon (Okinawa, Japan)

Outbreaks of the crown-of-thorns sea star (Acanthaster planci) are likely to be strongly associated with drastic changes in larval survival influenced by food availability. However, no quantitative or qualitative data are available on the distribution of A. planci larvae in the field nor on the environmental factors that influence their survivorship. Here we use a DNA barcoding approach to describe the distribution of A. planci larvae in Sekisei Lagoon, Ryukyu Archipelago, Japan after conducting three days of high-intensity sampling. High densities (53.3 individuals/m3) of A. planci larvae were found outside of Yonara Channel, which is the largest reef channel in this lagoon. Surprisingly, most (94%) of the aggregated larvae were advanced-stage brachiolaria. Considering that it takes several days to develop to this stage, this result demonstrates that A. planci larvae were floating for some time and maintaining a high-density population. However, this dense larval cloud disappeared immediately after a typhoon. No spatial correlation was found between larval density and either nutrient or chlorophyll a concentrations, suggesting that A. planci larvae do not necessarily aggregate in nutrient-rich water. These data suggest that some high-density populations of late developmental stage A. planci larvae were produced under a low phytoplankton concentration and could potentially trigger an adult outbreak. Consequently, our data suggest that adult outbreaks may not necessarily be triggered by food availability alone